Lidstrom:Measuring 13C Incorporation Into Protein - CO2 Project - Revision history

Amanda L Smith at 04:42, 28 December 2012

Amanda L Smith — Fri, 28 Dec 2012 04:42:07 GMT

←Older revision		Revision as of 04:42, 28 December 2012
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	== Mass Spec Reservation ==		== Mass Spec Reservation ==
-	* [http://pluto.chem.washington.edu/instrumentres/WeekSched.aspx?InstName=Agilent+5975+GCMS link]. It is $25/hour.	+	* [http://pluto.chem.washington.edu/instrumentres/WeekSched.aspx?InstName=Agilent+5975+GCMS link]. It is $25/hour. (last time, we had trouble editing the reservation after our time had started(

	== Cell Prep ==		== Cell Prep ==
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	== Derivitization of Amino Acids w/ TBDMS ==		== Derivitization of Amino Acids w/ TBDMS ==
	===Reagent Locations===		===Reagent Locations===
-	* TBDMS: ~~(???)~~	+	* TBDMS: Yanfen fridge door, although package says -20 deg C
-	* Internal standard: ~~(???)~~	+	* Internal standard: Box in Amanda's -20 deg C, need to make more
-	* pyridine:	+	* pyridine: Cabinet under fume hood
	=== Remove Ash ===		=== Remove Ash ===
	# Add 100 uL nanopure water to reconstitute the dried sample (if using 1 mL of OD600 = 0.6 culture)		# Add 100 uL nanopure water to reconstitute the dried sample (if using 1 mL of OD600 = 0.6 culture)
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	## use the same blue screw-cap but replace the septum		## use the same blue screw-cap but replace the septum
	# Rinse syringe in leftover pyridine (Yanfen does ten rinses)		# Rinse syringe in leftover pyridine (Yanfen does ten rinses)
-	##Repeat for all samples	+	## Repeat for all samples
	# Incubate for 1 hour in the heater closest to the RNA room door. Use the maximum temperature setting. Turn heater off after use. (should be 65<sup>o</sup>C, but was only 45<sup>o</sup>C on 11/19/2012, thermometer was broken, last reading 63-64 deg C 12/17/12)		# Incubate for 1 hour in the heater closest to the RNA room door. Use the maximum temperature setting. Turn heater off after use. (should be 65<sup>o</sup>C, but was only 45<sup>o</sup>C on 11/19/2012, thermometer was broken, last reading 63-64 deg C 12/17/12)

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	* Carrier gas = He		* Carrier gas = He
	* splitless injection (higher sensitivity than split-mode injection)		* splitless injection (higher sensitivity than split-mode injection)
-	* ~~needle~~ is washed in ~~hexane~~	+	* Needle is washed in hexanes
-	**more is available in room next door. Discard what is left in the HPlC waste jar, then refill bottle. Crack lid on squeeze tube so liquid doesn't get pushed out.	+
	* quadrupole is ceramic (??)		* quadrupole is ceramic (??)
	=== Instructions for Use ===		=== Instructions for Use ===
-	# Check that there is enough gas. Want at least 40-50 psi.	+	# Check that there is enough Carrier gas. Want at least 40-50 psi on the He tank.
-	# Check that there is enough ~~hexane~~ for needle washing.	+	# Check that there is enough hexanes for needle washing (Two vials should be in the A&B positions on the injection table)
-	##~~?? what is "enough"?~~	+	## Refill if near the min line
		+	###Refill the vials in the fumehood in the other room
		+	###Dump the old hexane in the waste container
		+	###Use the squeeze bottle in the fumehood to refill, if it's empty, there should be extra in the flammable cabinet to refill the squeeze bottle (loosen top after using the squeeze bottle)
	# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.		# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.
-	# Load method (name = ORGACD-TBDMS.M) by clicking on folder. Check that the settings are right. ~~Open~~ an old sample file, ~~found by opening~~ a ~~file~~ such as D:\Users\Amanda_Janet\2012_11_19-1st_13Cformate_test\5+48.D. Open the file acqmeth.txt	+	# Load method (name = ORGACD-TBDMS.M) by clicking on folder. Check that the settings are right by comparing to an old sample method file. To access an old sample file, find a sample folder such as D:\Users\Amanda_Janet\2012_11_19-1st_13Cformate_test\5+48.D. Open the file acqmeth.txt
-	## Click on thermometer & gc coil icon.	+	## Click on thermometer & gc coil icon. Go through the tabs and confirm the settings.
-	## ALS:	+	## ALS: Nothing
-	## ~~valves~~: don't set anything	+	## Valves: don't set anything
	## Inlets: septum purge flow: wash out that volume		## Inlets: septum purge flow: wash out that volume
	## Columns: flow is constant at 1 mL/min. Since temperature increases and He becomes more viscous at higher temps, the pressure ramps up, too. It is held high at the end to bake off anything that stuck.		## Columns: flow is constant at 1 mL/min. Since temperature increases and He becomes more viscous at higher temps, the pressure ramps up, too. It is held high at the end to bake off anything that stuck.
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	## Solvent delay: most of the sample is solvent. Let it run through.		## Solvent delay: most of the sample is solvent. Let it run through.
	## Gain Factor: Set to 1.00		## Gain Factor: Set to 1.00
-	## 1. 4 scans/~~seciond~~ --> enough data to draw a smooth curve	+	## 1. 4 scans/second --> enough data to draw a smooth curve
-	## Time ~~windo~~: how big the white window in the back looks	+	## Time window: how big the white window in the back looks
	## Zones: shows temps in mass spec. Don't want condensation		## Zones: shows temps in mass spec. Don't want condensation
-	## HiVac gauge: broke over weekend.	+	## HiVac gauge: sensor broke over weekend.
-	## Turbo Speed = 100 is desired. (green writing, black box, bottom right)	+	## Turbo Speed = 100 is desired. (green writing, black box, bottom right on the control panel)
		+	## HE output: 40 psi, 700 psi, worry at 2000 psi
	# File --> load method: chose default.M (prevents others from accidentally changing our method.)		# File --> load method: chose default.M (prevents others from accidentally changing our method.)
	# Load samples into rectangular rack. (count up as you go toward the back)		# Load samples into rectangular rack. (count up as you go toward the back)
	# Create file for sample run: will include the vial position and other useful info. (??)		# Create file for sample run: will include the vial position and other useful info. (??)
-	## ~~note~~: you can have it call your method from here. This will reduce the probability that other people will edit your method.	+	## Note: you can have it call your method from here. This will reduce the probability that other people will edit your method.
	## Open an existing file (optional)		## Open an existing file (optional)
-	## Update your sample names (no slashes!), vial position number, and copy the sample names into the data file names column.	+	## Update your sample names (no slashes!), vial position number, and copy the sample names into the data file names column (can't use key shortcuts for copy and paste).
-	## ~~make~~ sure the method for all rows is set to ours	+	## Make sure the method for all rows is set to ours
	## Make a new folder for where you want to store data (button at top left)		## Make a new folder for where you want to store data (button at top left)
	## Set method path at top, too (??)		## Set method path at top, too (??)
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	* Each sample gets it's own folder. There is a text file in each of these folders that tells the method used.		* Each sample gets it's own folder. There is a text file in each of these folders that tells the method used.
	* About our method:		* About our method:
-	** solvent washing: wash in A & B. Both are ~~hexane~~. There are also options to wash the needle with your sample or pump liquid in and out of the needle (this is a contamination risk.)	+	** solvent washing: wash in A & B. Both are hexanes. There are also options to wash the needle with your sample or pump liquid in and out of the needle (this is a contamination risk.)
	* Air shows up in mass spec.		* Air shows up in mass spec.
	** Can look for the air peaks to make sure it isn't leaking. M/Z is between 20 & 40.		** Can look for the air peaks to make sure it isn't leaking. M/Z is between 20 & 40.
-	* The machine can be calibrated with ~~___~~ that is held inside the machine. We can run these tests if we want, but Martin does somewhat regularly.	+	* The machine can be calibrated with standard that is held inside the machine. We can run these tests if we want, but Martin does somewhat regularly (~1x /week)

	== Supplies ==		== Supplies ==

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 19:22:53 GMT

Instructions for Use

←Older revision		Revision as of 19:22, 19 December 2012
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	# Check that there is enough memory by doing a sequence verification (square button: check mark)		# Check that there is enough memory by doing a sequence verification (square button: check mark)
	# Start by pressing run.		# Start by pressing run.
-	# Update the time on the GCMS reservation.	+	# Update the time on the GCMS reservation. 49 min/run so 1 hr is reasonable. It is probably more like 55.

	====Helfpul Info====		====Helfpul Info====

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 19:22:10 GMT

Instructions for Use

←Older revision		Revision as of 19:22, 19 December 2012
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	# Check that there is enough memory by doing a sequence verification (square button: check mark)		# Check that there is enough memory by doing a sequence verification (square button: check mark)
	# Start by pressing run.		# Start by pressing run.
		+	# Update the time on the GCMS reservation.

	====Helfpul Info====		====Helfpul Info====

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 19:20:57 GMT

Instructions for Use

←Older revision		Revision as of 19:20, 19 December 2012
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	## note: you can have it call your method from here. This will reduce the probability that other people will edit your method.		## note: you can have it call your method from here. This will reduce the probability that other people will edit your method.
	## Open an existing file (optional)		## Open an existing file (optional)
-	## Update your sample names, vial position number, and copy the sample names into the data file names column.	+	## Update your sample names (no slashes!), vial position number, and copy the sample names into the data file names column.
	## make sure the method for all rows is set to ours		## make sure the method for all rows is set to ours
	## Make a new folder for where you want to store data (button at top left)		## Make a new folder for where you want to store data (button at top left)

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 19:20:17 GMT

Instructions for Use

←Older revision		Revision as of 19:20, 19 December 2012
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	##?? what is "enough"?		##?? what is "enough"?
	# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.		# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.
-	# Load method (name = ORGACD-TBDMS.M) by clicking on folder. Check that the settings are right.	+	# Load method (name = ORGACD-TBDMS.M) by clicking on folder. Check that the settings are right. Open an old sample file, found by opening a file such as D:\Users\Amanda_Janet\2012_11_19-1st_13Cformate_test\5+48.D. Open the file acqmeth.txt
	## Click on thermometer & gc coil icon.		## Click on thermometer & gc coil icon.
	## ALS:		## ALS:

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 19:18:27 GMT

Instructions for Use

←Older revision		Revision as of 19:18, 19 December 2012
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	# Load samples into rectangular rack. (count up as you go toward the back)		# Load samples into rectangular rack. (count up as you go toward the back)
	# Create file for sample run: will include the vial position and other useful info. (??)		# Create file for sample run: will include the vial position and other useful info. (??)
-	## ~~You~~ can have it call your method from here. This will reduce the probability that other people will edit your method.	+	## note: you can have it call your method from here. This will reduce the probability that other people will edit your method.
-	## ~~Set path to method~~ and where you want to store data	+	## Open an existing file (optional)
-	## ~~Enter~~ sample ~~names~~.	+	## Update your sample names, vial position number, and copy the sample names into the data file names column.
-	# Start by ~~___~~.	+	## make sure the method for all rows is set to ours
		+	## Make a new folder for where you want to store data (button at top left)
		+	## Set method path at top, too (??)
		+	## Make sure you have an air blank as the 1st sample. (empty GCMS vial required)
		+	### Can set the sample type to blank or sample b/c not using their software.
		+	# Check that there is enough memory by doing a sequence verification (square button: check mark)
		+	# Start by pressing run.

	====Helfpul Info====		====Helfpul Info====

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 18:55:06 GMT

Instructions for Use

←Older revision		Revision as of 18:55, 19 December 2012
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	## HiVac gauge: broke over weekend.		## HiVac gauge: broke over weekend.
	## Turbo Speed = 100 is desired. (green writing, black box, bottom right)		## Turbo Speed = 100 is desired. (green writing, black box, bottom right)
		+	# File --> load method: chose default.M (prevents others from accidentally changing our method.)
	# Load samples into rectangular rack. (count up as you go toward the back)		# Load samples into rectangular rack. (count up as you go toward the back)
	# Create file for sample run: will include the vial position and other useful info. (??)		# Create file for sample run: will include the vial position and other useful info. (??)

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 18:53:55 GMT

Instructions for Use

←Older revision		Revision as of 18:53, 19 December 2012
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	## Zones: shows temps in mass spec. Don't want condensation		## Zones: shows temps in mass spec. Don't want condensation
	## HiVac gauge: broke over weekend.		## HiVac gauge: broke over weekend.
-	## Turbo Speed = 100 is desired.	+	## Turbo Speed = 100 is desired. (green writing, black box, bottom right)
	# Load samples into rectangular rack. (count up as you go toward the back)		# Load samples into rectangular rack. (count up as you go toward the back)
	# Create file for sample run: will include the vial position and other useful info. (??)		# Create file for sample run: will include the vial position and other useful info. (??)

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 18:46:23 GMT

Instructions for Use

←Older revision		Revision as of 18:46, 19 December 2012
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	##?? what is "enough"?		##?? what is "enough"?
	# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.		# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.
-	# Load method (name = ORGACD-TBDMS.M) by clicking on folder.	+	# Load method (name = ORGACD-TBDMS.M) by clicking on folder. Check that the settings are right.
		+	## Click on thermometer & gc coil icon.
	## ALS:		## ALS:
	## valves: don't set anything		## valves: don't set anything

Amanda L Smith: /* Instructions for Use */

Amanda L Smith — Wed, 19 Dec 2012 18:42:31 GMT

Instructions for Use

←Older revision		Revision as of 18:42, 19 December 2012
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	##?? what is "enough"?		##?? what is "enough"?
	# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.		# Open 579C/Enhanced to control machine. There is an icon on the desktop that may help the GCMS talk to the computer if they aren't already.
-	# ~~Check~~ method (name = ~~(?)~~ ORGACD-TBDMS)	+	# Load method (name = ORGACD-TBDMS.M) by clicking on folder.
	## ALS:		## ALS:
	## valves: don't set anything		## valves: don't set anything